Activity-Dependent Gene Expression in Human Epilepsy

描述（由申请人提供）：癫痫是一种常见但通常具有破坏性的神经系统疾病，其中异常的大脑兴奋导致无法控制和不可预测的正常功能丧失，通常以强直阵挛性癫痫发作结束。在大多数情况下，癫痫发作是在大脑的一个小区域开始的，没有明显的潜在遗传或结构缺陷。这种“癫痫灶”可以局部化并通过手术切除，以潜在地治愈癫痫发作。究竟是什么使和维持人类新皮层的某些区域癫痫是未知的，也不知道发生了什么结构和功能变化，导致和维持这种状态。一种越来越受到关注的可能性是，这些变化是通过生长和分化因子启动的途径中的活性依赖性变化而发生的。这些因子通过信号传导中间体来调节转录因子，转录因子打开基因，使大脑皮层过度兴奋。使用硬膜下记录电极映射人类新皮层癫痫灶在儿童顽固性癫痫发作，高通量筛选方法相结合的重点，高密度的cDNA阵列与广泛的搜索寡核苷酸阵列被用来识别基因差异表达的癫痫灶相比，附近地区没有独立的癫痫样活动。使用这些方法确定了两个重要的信号通路;一个由神经营养因子（BDNF）启动，另一个由神经调节蛋白启动。这些因子分别激活NFkB和ETS-2转录因子，进而调节已知调节神经元兴奋性的蛋白质。本文提出的研究将采用实时RT-PCR、原位杂交和免疫组织化学方法测量人类癫痫组织中神经营养素和神经调节素信号通路基因的差异表达，并将这些重要发现扩展到使用cDNA和寡核苷酸微阵列的更大系列患者。假设这些基因表达变化与电活动直接相关，并且将在癫痫发作扩散区域和沿着癫痫样活动的发作间期梯度中发现。然后，这些变化将诱导并维持组织的过度兴奋状态。了解调控基因的活性依赖性表达，并确定表达这些基因的细胞类型，将有助于了解癫痫发作在人类中的发展，这将有助于临床管理，并为癫痫患者揭示新的治疗途径。

DESCRIPTION (provided by applicant): Epilepsy is a common but often devastating neurological condition where abnormal brain excitation leads to uncontrolled and unpredictable loss of normal function often ending in a tonic-clonic seizure. In a majority of cases the seizures are initiated in a small region of the brain with no clear underlying genetic or structural defect. This "epileptic focus" can be localized and surgically removed to potentially cure the seizures. Exactly what makes and maintains certain areas of human neocortex epileptic is not known, nor is it known what structural and functional changes occur that lead to and maintain this state. One possibility that has gained increasing attention is that these changes come about through activity-dependent changes in pathways that are initiated through growth and differentiation factors. These factors act through signaling intermediates to regulate transcription factors that turn on genes that can make the cortex hyperexcitable. Using subdural recording electrodes to map human neocortical epileptic foci in children with intractable seizures, a high-throughput screening method that combines focused, high-density cDNA arrays with wide searching oligonucleotide arrays was used to identify genes differentially expressed at epileptic foci compared to nearby areas with no independent epileptiform activity. Using these methods two important signaling pathways were identified; one initiated by neurotrophins (BDNF) and the other by neuregulin. These factors activate the NFkB and ETS-2 transcription factors, respectively, which in turn regulate proteins known to modulate neuronal excitability. The studies proposed here will measure the differential expression of neurotrophin and neuregulin signaling pathway genes in human epileptic tissues using real-time RT-PCR, in situ hybridization, and immunohistochemistry and will extend these important findings to a larger series of patients using cDNA and oligonucleotide microarrays. The hypothesis is that these gene expression changes correlate directly with electrical activity and will be found in regions of seizure spread and along interictal gradients of epileptiform activity. These changes would then induce and maintain the hyperexcitable state of the tissue. Understanding activity-dependent expression of regulatory genes together with defining the cell types expressing these genes will provide insights into how seizures develop in humans that will aid in clinical management and reveal new therapeutic avenues for patients with epilepsy.